DESCRIPTION: Methods will be developed for extremely rapid (several minutes) characterization of picomole quantities of unknown proteins initially separated by one- or two-dimensional gel electrophoresis. This will be accomplished by elution of an protein, localized on a gel by staining, onto the surface of a time-of-flight mass spectrometer sample probe activated by covalent attachment of one or more digestive enzymes, typically endoproteases. These activated surfaces will be used to catalyze rapid digestion of the analyte, which may be followed by solution-phase exoprotease or chemical digestion to produce a ladder of fragments stemming from one or more of the endoprotease fragments. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the endoprotease digest fragments and of the mixture resulting from exoprotease digestion will yield an endoprotease map and a length of internal sequence for the initial protein which will be sufficient to identify it in a database search. Methods of protein localization and elution and the chemistries of digestion will be optimized and the limits of the technology in terms of protein size and chemistry, and sample amount, will be explored in collaborative studies.